The successful cloning and expression of cruzain facilitates mutagenesis, kinetics and crystallization experiments which will provide insights into structure, mechanism of catalysis, and function. The recombinant expression system provides active enzyme which can be used to test specific inhibitors. Furthermore, variant enzymes can be produced to determine the substrate preferences and details about the catalytic mechanism of the protease. This information will be useful for designing peptide inhibitors which show higher selectivity for the parasite protease than for the related mammalian proteases. These lead compounds could be further improved by analysis of the binding of the new inhibitors with recombinant cruzain in additional crystal structures. To anticipate mechanisms of resistance the parasite may mount against rationally designed drugs, PCR primers based on the gene sequence will be used to amplify and sequence mutant enzyme genes from resistant organisms derived from in vitro or in vivo screens. These altered genes could then be expressed and analyzed biochemically, and the position of the alterations mapped against known binding domains in the crystal structure. Cruzain contains an unusual carboxy terminal "trial." Several theories have been proposed for the function of this domain. Our successful expression of active recombinant enzyme has allowed us to initiate studies to analyze enzyme function and folding of expressed enzyme in mutants lacking the domain. We will now follow up on these studies with an experiment in which parasite cells, will be transfected with mutant versus wild type C-terminal domain ligated to a reporter gene. Our hypothesis is that the C-terminal domain is involved in trafficking or localization of the enzyme. Our ability to localize wild type protease by fluorescent probes will provide the background in which the phenotype of the transfected reporter constructs can be interpreted.